Growing evidence indicates that ribosome processivity during the translation of mRNA to proteins is exquisitely sensitive to changes in tRNA expression and processing. Perturbation of these processes contribute to aberrant cellular homeostasis that manifests as cancer or neurodegeneration. In a N-ethyl-N-nitrosourea (ENU) mutagenesis forward genetic screen in C57BL/6J (B6J) mice to identify novel genes regulating neuronal homeostasis, my lab identified a new ribosome rescue protein GTPBP2. Additionally, we identified a mutation in n-Tr20, a tRNAArgUCU that is specifically expressed in neurons, in B6J mice that epistatically interacts to cause neurodegeneration in Gtpbp2-/- mice. This mutation reduces the processing of n-Tr20 which in turn reduces the total pool of tRNAArgUCU in the brain causing ribosome stalling that is normally rescued by wild type GTPBP2. Interestingly, expression of n-Tr20 is restricted to the brain. These results established ribosome stalling as a novel mechanism for neurodegeneration and defined n-Tr20 as the first reported neuron-specific tRNA in vertebrates. Subsequent mapping crosses of B6J-Gtpbp-/- mice have identified a BALB/cByJ-derived locus that enhances neurodegeneration. Although we have narrowed the critical region containing this modifier gene, no causal gene or mechanism have been identified. In Aim 1 of this proposal, I have proposed a research strategy to identify the causative gene for this locus and elucidate the mechanisms by which this modifier gene augments neurodegeneration. In Aim 2, I will investigate the prevalence of cell-type tRNA expression in the brain by generating the first in vivo tRNA expression brain atlas. I will accomplish this aim by first generating a knock-in mouse line to conditionally epitope-tag RNA Polymerase III (Pol III), the RNA polymerase responsible for transcribing all tRNA, and then performing ChIP-sequencing to determine the occupancy of Pol III machinery on tRNA genes isolated from specific cell-types. With the completion of this proposal we expect to reveal the vast complexity of cell-type specific tRNA profiles in the mammalian brain, and new mechanisms of dysregulation that contribute to aberrant ribosome processivity and neuronal homeostasis in specific cell-types.